Oligonucleotides are utilized for a variety of biotechnological applications, based on their ability to confer specificity by virtue of their sequence composition. Given, for example, their ability to be designed to target a protein-encoding molecule, such as RNA, a particular use of oligonucleotides is in antisense technology.
Antisense Oligonucleotides (AONs)
Antisense oligonucleotides (AONs) have attracted considerable interest in the biotechnology sector, and have exceptional potential for use in therapeutic strategies against a range of human diseases. The formation of a duplex between the AON and its complementary sequence on its target (usually messenger RNA [mRNA]) prevents the translation of such RNA, in part by “translation arrest” (via duplex formation between the AON and the target RNA, thus inhibiting/preventing complete translation by physically or sterically blocking the translational machinery) but more importantly by eliciting degradation of the targeted RNA through the action of ribonuclease H(RNase H), a ubiquitous and endogenous cellular enzyme that specifically degrades the RNA strand in the AON/RNA duplex.
Since the natural substrate of RNase H is a DNA/RNA heteroduplex, DNA has been utilized for antisense technology. However, as serum and intracellular nucleases rapidly degrade AONs with phosphodiester (PDE) linkages, AON consisting of PDE-DNA have had limited utility in such systems. DNA with phosphorothioate linkages (PS-DNA) can induce RNase H degradation of the targeted RNA, and is resistant to degradation by serum and cellular nucleases, however, it forms weaker duplexes with the target RNA compared to PDE-DNA.
RNase H
RNase H selectively degrades the RNA strand of a DNA/RNA heteroduplex (Hausen, P.; Stein, H. Eur. J. Biochem. 1970, 14, 279). Studies with eukaryotic cell extracts containing RNase H suggest that both prokaryotic and eukaryotic enzymes exhibit similar RNA-cleavage properties (Monia et al. J. Biol. Chem. 1993, 268, 14514; Crooke et al. Biochem J. 1995, 312, 599; Lima, W. F.; Crooke, S. T. Biochemistry 1997, 36, 390). E. coli RNase H1 is thought to bind to the minor groove of the DNA/RNA double helix and to cleave the RNA by both endonuclease and processive 3′-to-5′ exonuclease activities (Nakamura, H. et al. Proc. Natl. Acad. Sci. USA 1991, 88, 11535; Fedoroff, O. Y. et al., J. Mol. Biol. 1993, 233, 509). The efficiency of RNase H degradation displays minimal sequence dependence and, as mentioned above, is quite sensitive to chemical changes in the antisense oligonucleotide.
There is therefore a need for an improved oligonucleotide, to address one or more of the limitations noted above.